Detection of human immunodeficiency virus

ABSTRACT

The invention relates to a method of detecting a HIV-infected cell in a mammal. The method includes detecting an HIV 2-LTR circle DNA molecule obtained from a cell of an HIV-positive mammal, especially an HIV-1-positive human.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional PatentApplication Serial No. 60/115,228, filed Jan. 8, 1999, now abandoned.

STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH

[0002] The invention was made with Government grants from the NationalInstitutes of Health (RR11589, HL57880, AI32391, and AI32907). TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The invention relates to human immunodeficiency virus (HIV)detection assays.

BACKGROUND OF THE INVENTION

[0004] Various assays have been developed to detect HIV. A common HIV-1detection assay utilizes quantitative polymerase chain reaction (PCR) asa means to amplify and detect viral RNA present in patient plasma. HIV-1positive individuals undergoing combination antiviral therapy (i.e.,receiving two or more anti-HIV-1 compounds) can exhibit decreased viralloads in the peripheral blood. In some cases, after several weeks ormonths of therapy, HIV-1 RNA cannot be detected in the peripheral blood,indicating possible eradication of HIV-1 in those individuals.Unfortunately, if patients exhibiting such a seemingly negative resultstop therapy in the face of continued viral replication below thesensitivity of detection assays, the HIV can rebound very rapidly. Thus,the limited sensitivity of HIV detection assays provides a challenge tofurther advances in therapy.

SUMMARY OF THE INVENTION

[0005] The invention is based on the discovery that patients having novirus detectable in the blood by known means, e.g., patients undergoingdrug therapy, such as combination drug therapy, can nevertheless harbornewly HIV-infected cells, and that these newly infected cells can bedetected by the presence of 2-LTR circles in peripheral bloodmononuclear cells. The presence of the 2-LTR circles indicates thepersistence of viral spread in those patients, in spite of the fact thatthese patients have no detectable virus in the blood.

[0006] The invention is also based on the discovery that, for otherpreviously HIV-positive patients without detectable virus, no 2-LTRcircles are present, indicating that eradication of HIV is possible.

[0007] Accordingly, the invention features a method of detecting anHIV-infected cell (e.g., an HIV-1-infected cell) in a mammal (e.g., ahuman) undergoing combination anti-HIV drug therapy by detecting an HIV2-LTR circle DNA molecule obtained from a cell (e.g., a peripheral bloodmononuclear cell) of the mammal. In one embodiment of the method,cell-free HIV viral RNA cannot be detected in the blood of the mammal.

[0008] In another aspect, the invention features a method of detectingan HIV-infected cell (e.g., an HIV-1-infected cell) in a mammal (e.g., ahuman) by detecting an HIV 2-LTR circle DNA molecule obtained from acell (e.g., a peripheral blood mononuclear cell) of an HIV-positivemammal (e.g., an HIV-1-positive human). In this method, cell-free HIVviral RNA cannot be detected in the blood of the mammal. In oneembodiment, the mammal undergoes combination anti-HIV drug therapy.

[0009] The invention also includes a method of treatment for HIVinfection in a mammal (e.g., a human) by (1) administering to the mammal(e.g., a human) one or more anti-HIV agents (e.g., one or more HIVprotease inhibitors and one or more HIV reverse transcriptaseinhibitors) in an amount effective to reduce an HIV viral load (e.g.,circulating plasma viral load) in the mammal; and (2) detectingHIV-infected cells in the mammal using the detection methods of theinvention. The treatment is continued until the level of HIV-infectedcells falls below 100 (e.g., below 50, 25, 10, 5 or 1) in one millionperipheral blood mononuclear cells (PBMC).

[0010] In the methods of the invention, the HIV 2-LTR circle DNAmolecule can be obtained from the cell using an alkaline lysis method.In addition, the detecting step can include amplifying (e.g., by PCR)the DNA molecule before the detecting step. Examples of primers used forPCR include a (−) strand primer spanning nucleotides 9591 to 9610 of theHXB2 strain of HIV-1, and a (+) strand primer spanning nucleotides9650-9669 of the HXB2 strain of HIV-1.

[0011] A combination anti-HIV drug therapy includes the administrationof two or more antiviral compounds. Such compounds include HIV reversetranscriptase inhibitors such as zidovudine (ZDV), didanosine (ddI), andzalcitabine (ddC); and HIV protease inhibitors such as indinavir (IDV),ritonavir (RTV), saquinavir (SQV), and nelfinavir (NFV). Other compoundsinclude inhibitors of integration, virus assembly, envelope glycoproteinbinding to receptor or co-receptor, or fusion between virus and cellmembranes.

[0012] An HIV-positive individual is one who produces antibodies thatspecifically bind to an HIV viral protein.

[0013] Cell-free HIV viral RNA is RNA that is not associated with a cell(e.g., RNA in plasma).

[0014] The methods of the invention are useful for evaluating the levelof newly HIV-infected cells in HIV-positive individuals undergoingantiviral therapy. A feature of the methods is the detection of 2-LTRcircles. Since 2-LTR circles exist only in the first several hours afterproductive infection and are degraded thereafter, they are a suitablemarker for newly infected cells.

[0015] Confirmation of the eradication of HIV by the methods of theinvention gives the individual or his healthcare provider the option tocease antiviral therapy. Conversely, the presence of newly infectedcells during therapy, in spite of the absence of HIV RNA in the blood,signals to the healthcare provider to continue therapy. This isimportant because the premature cessation of therapy allows the virus togrow anew, often establishing a more persistent infection and moredevastating symptoms in the patient than before antiviral therapycommenced.

[0016] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although suitablemethods and materials for the practice or testing of the presentinvention are described below, other methods and materials similar orequivalent to those described herein, which are well known in the art,can also be used. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

[0017] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIGS. 1A and 1B are graphs of viral DNA (copies/10⁵ cells) versustime post RT inhibition, showing data for HIV-1_(LAI) and HIV-1_(ADA),respectively.

[0019] FIGS. 2A-2D are graphs of HIV-1 RNA or genomes versus time inweeks, showing the data for patients Gu, Sm, Za, and Ha, respectively.

[0020]FIG. 3 is a data point plot of number of virus-positive andvirus-negative cultures for patient designations.

DETAILED DESCRIPTION

[0021] The invention relates to a method of determining the level ofnewly HIV-infected cells (i.e., viral spread) in HIV-positive patients,for example, patients undergoing antiviral drug therapy, by detectingHIV 2-LTR circles in the patients' cells. The method is especiallyuseful for detecting HIV 2-LTR circles in patients testing negative forplasma viral RNA. As discussed above, patients without detectable plasmaviral RNA may still retain circulating virus. Thus, complete eradicationof virus from an HIV-positive individual should be confirmed by anabsence of newly HIV-infected cells in that individual. The methods ofthe invention provide a means for that confirmation.

[0022] In vitro studies of retroviruses have shown that the firstevidence of reverse transcription is unintegrated viral DNA appearing inthe cytoplasm, which is transported to the nucleus within hours afterinfection of a cell (Shank et al., J. Virol., 25:104-114, 1978; Claymanet al., Science, 206:582-584, 1979; and Stevenson et al., EMBO J.,9:1551-1560, 1990). In the case of HIV-1, this unintegrated DNA existsin several forms, including incompletely or completelyreverse-transcribed linear DNA, circular DNA containing one LTR, andcircular DNA containing two LTRs (2-LTR circles). 2-LTR circles areidentical to integrated proviruses, except that the ends of the LTR arejoined in a head-to-tail fashion via a linker sequence of a fewnucleotides.

[0023] As such, PCR can be used to specifically amplify a small segment(a few hundred base pairs) spanning the 2-LTR junction. The PCR will bespecific for 2-LTR circles, since neither proviruses, single LTRcircles, or other incomplete viral reverse transcription products willbe amplified.

[0024] Sample Preparation

[0025] A variety of biological samples can be analyzed by the methods ofthe invention, including blood and solid-tissue biopsies (e.g., a lymphnode biopsy). For example, blood can be collected from an HIV-positiveindividual undergoing combination therapy. PBMC are isolated by standardficoll-based isolation procedures. The PBMC are then lysed and the totalor extrachromosomal DNA isolated.

[0026] Total cellular DNA can be extracted by lysing the PBMC indetergent, digesting the cellular protein, and precipitating the DNA(Pauza et al., Virology, 205:470-478, 1984; and Panther et al., J.Acquir. Immune. Defic. Syndr. Hum. Retro., 17:303-313, 1998).Extrachromosomal DNA can be isolated by any method known in the art,including standard alkaline lysis, Hirt extraction, or guanidiniumthiocyanate precipitation (Jurrians et al., J. Gen. Virol.,73:1537-1541, 1992; Stevenson et al., J. Virol. 64:2421-2425, 1990; andSambrook et al., eds., Molecular Cloning: A Laboratory Manual. 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

[0027] Although the standard alkaline lysis technique is best known forisolating plasmid DNA from bacteria, this technique can also be usedaccording to the invention to isolate 2-LTR circles from mammaliancells. The Spin Miniprep Kit available from Qiagen (Cat. No. 27104) isespecially useful for this purpose. The methods of the invention includethe use of this technique to isolate and purify 2-LTR circle DNA.

[0028] When possible, extrachromosomal DNA, instead of total DNA, shouldbe isolated since the number of target 2-LTR circles per microgram ofextrachromosomal DNA is expected to be far greater than the number of2-LTR circles per microgram of total cellular DNA.

[0029] Detecting 2-LTR Circles

[0030] 2-LTR circles can be detected by standard techniques which do notrequire nucleic acid amplification, such as Southern blotting. The DNAsample obtained as described herein can be hybridized with 2-LTRcircle-specific probes that are directly or indirectly labeled withchromogenic, radioactive, fluorescent, or luminescent labels.

[0031] Where amplification of the 2-LTR circles before detection isdesired, the 2-LTR circles can be detected by any amplification methodwell known in the art. These methods include polymerase chain reaction(PCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202) and variants thereof.Another suitable nucleic acid amplification method is ligation chainreaction (LCR) or variants thereof (Landegran et al., Science,241:1077-1080, 1988; and Nakazawa et al., Proc. Natl. Acad. Sci. USA,91:360-364, 1994).

[0032] Other amplification methods include: self-sustained sequencereplication (Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874-1878,1990), transcriptional amplification system (Kwoh, et al., Proc. Natl.Acad. Sci. USA, 86:1173-1177, 1989), and Q-Beta Replicase (Lizardi etal., Bio/Technology, 6:1197, 1988).

[0033] However the 2-LTR circles are detected, a threshold level of2-LTR circles per million cells would be useful to define meaningfulnumbers of the circles. If the assay is capable of single-moleculesensitivity, a base threshold can be established at one circle permillion PBMC, which is appropriate when determining whether eradicationof HIV has been achieved in a patient. Whenever a patient tests abovethis threshold, the patient is said to exhibit active viral infection.Whenever a patient tests below the threshold, the patient is said to notexhibit active viral infection and is a candidate for removal fromantiviral therapy. In other contexts, such as when the level of 2-LTRcircles are used to determine the efficacy of any antiviral regiment,thresholds above one per million PBMC may be appropriate (e.g., 10, 50,100, or 250 circles/10⁶ PBMC).

[0034] Any of the above methods can be combined in a method of theinvention to achieve suitable 2-LTR detection efficiencies.

[0035] Administration of Antiviral Drugs

[0036] The dosages, specific formulations, and routes of administrationof HIV antiviral drugs are known in the art. See, eg., Physicians' DeskReference, Fifty-fourth edition, Medical Economics Company, Montvale,N.J., 2000.

[0037] The invention will be further described in the following example,which does not limit the scope of the invention described in the claims.

EXAMPLE 1

[0038] Blood samples were obtained using standard techniques from 20HIV-1-infected individuals who began and continued to receivecombination anti-HIV drug therapy. All of these patients exhibited aperiod of time in which, after commencement of combination therapy, noplasma viral RNA could be detected by quantitative PCR. PBMC wereisolated from each blood sample, and the extrachromosomal DNA waspurified using the Spin Miniprep Kit available from Qiagen as Cat. No.27104, generally following the manufacturer's directions.

[0039] HIV-1 2-LTR circles were detected by quantitative PCR using the2-LTR-specific primers described in Stevenson et al., J. Virol.,64:2421-2425 (1990). The (−) strand primer spanned nucleotides 9591 to9610 (or 507-526) of the HXB2 strain of HIV-1, while the (+) strandprimer spanned nucleotides 9650-9669 (or 566-585) of the HXB2 strain ofHIV-1 (Ratner et al., Nature, 313:277-284, 1985). Plasma viral RNA ineach sample was also measured using the Amplicor HIV Monitor™ kit (RocheMolecular Systems, Inc., Branchburg, N.J.), employing HIV-1-specificquantitative PCR, following manufacturer's directions. The threshold ofdetection for this standard HIV-1 RNA detection assay was about 50 viralRNA molecules per milliliter of plasma. On the other hand, the thresholdfor the method of the invention at which the number of 2-LTR circles wasconservatively estimated to give a positive result was set at 1 moleculeor circle per million PBMC (roughly about 0.1 to 1 ml whole blood).Higher thresholds could be set, but such thresholds may lead to morefalse negatives. Considering the consequences of false negatives, thelowest practical threshold should be used. The results are summarized inTable 1. TABLE 1 # Viral RNA/ # 2-LTR Circles/ Months without Patient mlPlasma 10⁶ cells Detectable Viral RNA 1 <50 20 N/A 2 155 25 8 3 <50 4712 4 <50 872 9 5 N/A 13 N/A 6 <50 <1 9 7 <50 6200 7 8 <50 <1 17 9 N/A 1N/A 10 <50 1 N/A 11 <50 240 19 12 121 36 19 13 N/A 3 15 14 <50 48 10 15<50 <1 24 16 <50 1 9 17 <50 3 15 18 <50 <1 16 19 <50 271 15 20 69 117 4

[0040] Table 1 illustrates the unexpectedly superior sensitivity of2-LTR circle detection as compared to the standard plasma viral loadassay. Some HIV-positive individuals, who do not have detectable plasmavirus, nevertheless harbor newly HIV-infected blood cells, as indicatedby the presence of 2-LTR circles (i.e., patients 1-5, 7, 9-14, 16, 17,19, and 20). These individuals should not cease antiviral therapy, sinceinfectious virus is still present in the body.

[0041] On the other hand, in some patients with undetectable plasmavirus, no 2-LTR circles were detected in their PBMC (i.e., patients 6,8, 15, and 18). These individuals may have completely eradicated HIVfrom their bodies, and are candidates for removal from antiviraltherapy.

EXAMPLE 2

[0042] The stability of 2-LTR circle forms of viral DNA were initiallyexamined in acutely infected cells in vitro. CD4⁺ MT-4 T cells andJurkat-CCR5 cells were infected with the X4 variant HIV-1_(LAI) and theR5 variant HIV-1_(ADA), respectively. Synthesis of viral cDNA wasallowed to proceed for 24 hours, and further rounds of virus infectionand cDNA synthesis were then restricted by the addition of reversetranscriptase inhibitors ZDV (5 μM) or Nevirapine (1 μM) to HIV-1_(LAI)and HIV-1_(ADA) infected cells, respectively. Cells were then maintainedin the presence of the RT inhibitors.

[0043] The experimental procedures used in Examples 2-4 are brieflydescribed.

[0044] The relationship between 2-LTR circle frequency and either theduration of undetectable plasma viral RNA or the frequency of positivevirus co-cultures was examined using Spearman's correlation coefficient.Mean frequency of positive co-cultures in 2-LTR circle positiveindividuals and 2-LTR circle negative individuals as shown in FIG. 3 wasfurther compared by a paired t-test.

[0045] Ficoll-purified PBMC (2-40×10⁶) were collected by centrifugationat 1300×g for 2 minutes. Cell pellets were resuspended in buffer P1 andextrachromosomal DNA was purified by a QIAprep™ spin miniprep kit(Qiagen, Valencia, Calif.) using the modification for the isolation oflow copy number plasmids as recommended by the manufacturer. ChromosomalDNA was recovered from the sodium acetate-SDS precipitate using DNAzol™reagent (Life Technologies, Gaithersburg, Md.) according to themanufacturer's protocol. Total cellular DNA was purified using anIsoquick™ nucleic acid extraction kit (ORCA Research, Bothell, Wash.).

[0046] 2-LTR circle junctions were amplified from 10-30 μl ofextrachromosomal DNA in a 50 μl reaction containing 1×HotStarTaq™buffer, 200 nM dNTPs, 400 nM primers, and 1.5 units HotStarTaq™ (Qiagen,Valencia, Calif.). The reverse primer was 5′-cagatctggtctaaccagaga-3′(SEQ ID NO:1), and the forward primer was 5′-gtaactagagatccctcagac-3′(SEQ ID NO:2), which annealed to nucleotides 9157-9137 (HIV-1 LTR Rregion) and nucleotides 130-150 (HIV-1 LTR U5 region) of HIV-1_(LAI),respectively (see GenBank Accession No. K02013 for numbering). After aninitial denaturation step (95° C., 10 minutes), PCR amplificationproceeded for 45 cycles (95° C., 30 seconds; 60° C., 30 seconds; 72° C.,60 seconds) followed by a final extension (72° C., 5 minutes).

[0047] To control for the effect of sequence polymorphisms at primerbinding sites, amplification was performed with internal primers whichwere reversed in orientation to those listed above. Amplification withthe internal LTR primers proceeded for 35 cycles using conditionsoutlined above. Polymorphisms in the region of the LTR that isrecognized by the fluorogenic probe can affect annealing of the probeand potentially result in “false negatives.” Consequently, Taqmanreaction products were subsequently analyzed on agarose-TBE gels andstained with ethidium bromide to ensure that those reactions did notcontain episome-specific PCR products. For quantitation of 2-LTR circlefrequency in patient PBMC, PCR reactions were performed using an ABIprism 7700 sequence detection system with the addition of 200 nMfluorogenic probe (5′-agtggcgagccctcagatgctgc-3′; SEQ ID NO:3) to thereaction. The probe anneals to nucleotides 9081-9103 of HIV-1_(LAI) andwas modified with 6-FAM (6-carboxyfluorescein) reporter dye on the 5′end and 6-TAMRA (6-carboxytetramethylrhodamine) quencher dye on the 3′end. Copy number estimates of 2-LTR circles were determined byextrapolation from a plot of standards versus band intensity or by usingthe ABI prism 7700 quantitation software. For sequencing, 2-LTR circlejunctions were cloned into a TA cloning vector (Invitrogen, San Diego,Calif.) and analyzed on an ABI 377 DNA sequencer according to themanufacturer's protocol.

[0048] Patient PBMC were separated by Ficoll-Paque (Amersham-Pharmacia)and depleted of CD8⁺ T lymphocytes using antibody-coated beads (Dyna1).Cells were seeded in flasks in aliquots of 1×10⁷ cells in RPMI 1640medium supplemented with 10% fetal calf serum and activated by PHA (5μg/ml) for 12 hours. CD8⁺-depleted PBMC from HIV-1 seronegativeindividuals were activated for 12 hours with PHA and added in equalnumbers to flasks of patient PBMC together with 20 IU/ml ofinterleukin-2 (Genzyme). At weekly intervals, half of the culturesupernatant was replaced with fresh medium containing 20 IU/ml IL-2 and10⁷ freshly isolated, CD8⁺-depleted, PHA activated donor PBMC from HIV-1seronegative individuals. HIV-1 Gag p24 antigen in culture supernatantswas evaluated by ELISA (Beckman Coulter) after 4 weeks.

[0049] Within 24-48 hours following addition of the RT inhibitors, 2-LTRcircle number fell by over ten fold in both HIV-1_(LAI) and HIV-1_(ADA)infected cells (FIGS. 1A and 1B). The copy number of other viral DNAforms identified by the internal LTR primers (predominantly linear andintegrated viral genomes) remained relatively constant over the sameinterval. Thus, 2-LTR circles appeared to be labile intermediates in thevirus lifecycle.

EXAMPLE 3

[0050] Whether 2-LTR circles were labile in vivo was next evaluated.PBMC samples were obtained from four HIV-1 infected individuals (Gu, Sm,Za, Ha) who, following adjustment of their antiretroviral regimens tomore potent combinations, exhibited steady declines in plasma viral RNAlevels. Patient Gu, who had been maintained on a two-drug RT inhibitorcombination, was subsequently changed (week 0) to a three-drug regimen(ZDV/3TC/NFV). Patient Sm, who had been on a two-drug regimen (ZDV/3TC)was changed at week 68 to ddI/EFV/NFV. Patient Za, who had been on afour-drug regimen (3TC/D4T/ddI/NFV) was adjusted (week 1) toZDV/ddC/NFV/RTV. Patient Ha, previously on a three-drug regimen(ZDV/ddI/NVP) was subsequently adjusted (week 0) to D4T/3TC/NVP. Markeddeclines in 2-LTR circle copy number were observed over the interval inwhich there was a rapid drop in levels of plasma viral RNA (FIGS.2A-2D). In contrast, when samples were analyzed in parallel withinternal LTR primers, HIV-1 viral genome levels (detected via cDNA)fluctuated by no more than three fold (FIGS. 2A-2C). Collectively, theresults suggest that 2-LTR circles are labile, both in vitro and invivo, relative to integrated viral genomes.

EXAMPLE 4

[0051] A larger patient population than that of Example 1 was thenexamined. The 2-LTR HIV-1 episomes were examined in 63 patients (four ofwhom were included in the study described in Example 1) who, throughtreatment with high activity antiretroviral therapy (HAART), hadundetectable levels of plasma viral RNA for sustained periods of time(Table 2). Fifty of these patients (80%) had undetectable levels ofplasma viral RNA (assay limit of sensitivity was 400 copies/ml) for 12months or longer (Table 2). Of these 50 patients, 24 (48%) exhibitedundetectable levels of plasma viral RNA for 12 months or more using anassay with a sensitivity of 50 copies/ml. In 48 of the 63 patients(76%), 2-LTR circles were detected in their PBMC (Table 2). 2-LTR circlecopy numbers ranged from less than 1 copy/10⁶ PBMC to 620 copies/10⁶PBMC. There did not appear to be any significant relationship betweenthe frequency of 2-LTR circles in patient PBMC and the time during whichplasma viral RNA was undetectable. This data indicated that labilereplication intermediates are present in a substantial proportion ofHIV-1 infected individuals who exhibit sustained suppression of plasmaviral RNA while on HAART. 2-LTR circles were not detectable in PBMC from15 (24%) patients (Table 2).

[0052] Table 2 below lists AIDS patients on HAART and the level of 2-LTRcircles and viral RNA in the blood. The abbreviations for Table 2 are asfollows. Anti-retroviral therapy: ZDV, Zidovudine; 3TC, Lamivudine; D4T,Stavudine; dd1, Didanosine; NVP, Nevirapine; RTV, Ritonavir; EFV,Efavirenz; SQV, Saquinavir; IDV, Indinavir; NFV, Nelfinavir; ddC,Zalcitabine; and ABV, Abacavir. CD4⁺ T cell measurements were determinedat or just prior to time PBMC were collected for PCR analysis of viralcDNA intermediates.

[0053] For the column labeled “Period of Undetectable Viral RNA,” plasmaviral RNA was detected using an assay with a sensitivity of about 400copies/ml. Numbers in parentheses indicated the period for which viralRNA was below the level of detection using a second assay with asensitivity of 50 copies/ml. Plasma viral RNA measurements weredetermined approximately every three months.

[0054] The 2-LTR circle copy number in most cases were determined induplicate on independent PBMC samples. Values less than 1 indicated thatmore than 1 million PBMC were required for detection of 2-LTR circles.

[0055] The total number of PBMC from which extrachromosomal DNA wasisolated and analyzed for the presence of 2-LTR circles was determinedas follows. In all patients, 2-LTR circles were quantitated byfluorescence-based PCR using Taqman software (ABI Prism 7700 Software).Similar 2-LTR circle numbers were obtained when samples were quantitatedby comparison of PCR band intensity to a standard dilution of synthetic2-LTR circles. TABLE 2 Period of CD4⁺ T Undetectable Patient Cells ViralDNA 2-LTR circles #PMBC Analyzed Number Drug Regiments (cells/ml)(months) (Copies/10⁶ PMBC) (millions) W 1 RTV, ZDV, 3TC 475 23 (14) 31.0 W 2 NFV, ZDV, 3TC 827 13 (13) <1 5.5 W 3 IDV, D4T, 3TC 436 23 (14)27 1.0 W 4 IDV, D4T, 3TC 505 22 (12) 37 1.0 W 6 IDV, D4T, 3TC 248 19(11) 15 1.0 W 7 SQV, D4T, 3TC 443 19 (13) 8 1.0 W 8 ddl, D4T 870 18 (15)<1 4.0 W 9 NFV, D4T, 3TC 641 22 (11) 59 1.0 W10 IDV, ZDV, 3TC 656 22(15) <1 4.0 W11 IDV, ZDV, 3TC 344 22 (15) 65 1.0 W12 ZDV, 3TC, DLV 62626 (16) <1 5.5 W13 NFV, ZDV, 3TC 699 13 (13) <1 5.5 W14 NFV, D4T, 3TC685 21 (15) 47 1.0 W15 NFV, STC, NVP 866 25 (12) 17 1.0 W16 RTV, D4T,3TC 572 22 (14) 2 5.5 W17 IDV, ZDV, 3TC 364 26 (15) 31 1.0 W18 IDV, ZDV,3TC 119 21 (16) <1 2.0 W19 SQV, ZDV, 3TC 153 16 (10) 4 4.0 W20 IDV, ZDV,3TC 360 27 (15) <1 4.0 W21 NFV, D4T, 3TC 208 13 (13) <1 2.0 W22 D4T, 3TC495 23 (15) <1 4.0 W28 NFV, ddl, D4T 527 22 (8) 9 1.0 W30 D4T, 3TC 57522 (17) <1 4.0 M 1 NFV, D4T, NVP 287 14 (9) 31 1.0 M 3 LDV, ddl, NVP 44016 (7) 22 1.0 M 4 IDV, ZDV, 3TC 586 13 (ND) 264 1.0 M 6 NFV, ZDV, 3TC317 24 (7) 63 1.0 M 7 NFV, 3TC, NVP 175 11 (ND) 4 5.5 M 8 IDV, ZDV,3TC,NVP 357 13 (2) 15 1.0 M12 NFV, D4T, 3TC 749 12 (7) 35 1.0 M13 ZDV,3TC, EFV 670 10 (0) 67 1.0 M14 IDV, ZDV, 3TC 728 14 (14) 41 1.0 M15 IDV,ZDV, 3TC 565 10 (10) 82 1.0 M16 NFV, 3TC, NVP 403 12 (8) 3 4.0 L 2 3TC,D4T, RTV 852 8 (8) 5 1.0 L 3 ZDV, 3TC, IDV 448 12 (8) 10 1.0 L 4 ZDV,3TC, RTV 978 21 (12) 180 1.0 L 6 D4T, RTV, SQV 577 10 (7) <1 4.0 L 7D4T, ddl, NVP 394 11 (7) 610 1.0 L 8 ZDV, 3TC, NFV 173 17 (8) <1 1.0 L 93TC, D4T, EFV 482 8 (5) <1 2.2 L11 ZDV, 3TC, RTV 615 19 (12) 84 1.0 L123TC, D4T, RTV 389 19 (6) 7 1.0 L13 D4T, SQV, NFV 312 15 (3) <1 7.8 L143TC, D4T, IDV 375 14 (7) 116 1.0 L15 3TC, RTV, SQV, ABV 91 30 (17) <11.5 L16 3TC, D4T, SQV, RTV 575 12 (12) 4 8.1 L17 3TC, D4T, SQV 198 15(15) 14 1.0 L18 ZDV, 3TC, IDV 175 16 (13) <1 10.2 L19 3TC, D4T, RTV, SQV499 15 (6) 620 1.0 L22 ZDV, D4T, LDV 223 14 (12) 6 1.0 L23 3TC, ddC, IDV534 14 (12) <1 4.8 L26 3TC, D4T, SQV, NIFV 911 17 (6) 36 1.0 L27 ZDV,3TC, IDV 185 17 (17) <1 3.2 L28 D4T, ABV, EFV 80 8 (8) 275 1.0 L29 ZDV,ddC, SQV, NFV 121 21 (1) 3 2.0 L32 3TC, D4T, EFV 219 7 (1) <1 10.0 L333TC, D4T, IDV 610 16 (1) <1 14.4 L36 ddl, D4T, NFV 172 14 (4) 2 5.6 L37ZDV, ddC, 3TC, IDV 279 13 (7) <1 5.6 L41 ZDV, 3TC, RTV 990 22 (1) 1001.0 L42 3TC, D4T, SQV 117 18 (1) <1 2.0 L46 3TC, D4T, NFV 180 7 (1) 420.0

[0056] It was suspected that in 2-LTR circle positive patients, therewould also be cells harboring replication competent virus. Toinvestigate this, high-input viral co-culture assays were performed onPBMC from nine 2-LTR circle positive and four 2-LTR circle negativepatients. The results are shown in FIG. 3. Replication competent viruscould readily be isolated from eight of the nine patients who were 2-LTRcircle positive. Virus could not be isolated from patient W1 who had avery low circle copy number. Intriguingly, infectious virus could not beisolated from three patients who were 2-LTR circle negative even thoughco-culture was conducted on between 40 and 60 million CD8+-depletedpatient PBMC. In patient L8 who was also 2-LTR circle negative, only oneof three cultures yielded infectious virus (FIG. 3). Collectively, theseresults suggested a correlation between the presence of 2-LTR circlesand cells harboring replication competent virus. Plasma based viral RNAassays therefore, unlike the 2-LTR circle assay, failed to reveal thefull extent of viral activity in infected individuals who are beingtreated with HAART.

[0057] This study has important implications for the development ofstrategies to eradicate virus replication in HIV-1 infected individuals.Although complete elimination of HIV-1 replication may be difficult withcurrent antiretroviral regimens, this study suggests instances in whicheven the most sensitive assays fail to reveal ongoing replication insome well suppressed patients. It is also likely that, as more potentantiretrovirals enter the clinic, ongoing or “covert” virus replicationmay be arrested in a higher percentage of patients. A betterunderstanding of the nature of the reservoir which sustains virusreplication in aviremic patients on HAART may lead to the development ofmore effective strategies for arrest of virus replication. Monitoring ofthe 2-LTR, as a superior surrogate marker for viral replication, can beintegral to the understanding of viral reservoirs.

Other Embodiments

[0058] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of detecting an HIV-infected cell from amammal undergoing combination anti-HIV drug therapy, the methodcomprising detecting an HIV 2-LTR circle DNA molecule obtained from acell of the mammal, wherein the presence of a 2-LTR circle DNA indicatesan HIV-infected cell.
 2. The method of claim 1, further comprisingamplifying the DNA molecule before the detecting step.
 3. The method ofclaim 2, wherein the DNA molecule is amplified using polymerase chainreaction.
 4. The method of claim 1, wherein the drug therapy comprisesadministering to the mammal at least one HIV reverse transcriptaseinhibitor.
 5. The method of claim 4, wherein the drug therapy furthercomprises administering to the mammal at least one HIV proteaseinhibitor.
 6. The method of claim 1, wherein the drug therapy comprisesadministering to the mammal at least one HIV protease inhibitor.
 7. Themethod of claim 1, wherein the mammal is an HIV-1-positive mammal. 8.The method of claim 7, wherein the mammal is a human.
 9. The method ofclaim 1, wherein the mammal is a human.
 10. The method of claim 1,wherein the cell is a peripheral blood mononuclear cell.
 11. The methodof claim 1, wherein cell-free HIV viral RNA cannot be detected in theblood of the mammal.
 12. A method of detecting an HIV-infected cell in amammal, the method comprising detecting an HIV 2-LTR circle DNA moleculeobtained from a cell of a mammal, wherein cell-free HIV viral RNA cannotbe detected in the blood of the mammal, and wherein the presence of a2-LTR circle DNA indicates a HIV-infected cell.
 13. The method of claim12, further comprising amplifying the DNA molecule before the detectingstep.
 14. The method of claim 13, wherein the DNA molecule is amplifiedusing polymerase chain reaction.
 15. The method of claim 12, wherein themammal is an HIV-1-positive mammal.
 16. The method of claim 15, whereinthe mammal is a human.
 17. The method of claim 12, wherein the mammal isa human.
 18. The method of claim 12, wherein the cell is a peripheralblood mononuclear cell.
 19. A method of detecting an HIV-1-infectedperipheral blood mononuclear cell (PBMC) in an individual, the methodcomprising amplifying an HIV-1 2-LTR circle DNA molecule obtained from aPBMC of an HIV-1-positive individual undergoing combination anti-HIV-1drug therapy, to produce an amplified nucleic acid, wherein cell-freeHIV-1 viral RNA cannot be detected in the blood of the individual; anddetecting the amplified nucleic acid, wherein the presence of theamplified nucleic acid indicates the presence an HIV-infected PBMC. 20.A method of claim 1, further comprising obtaining the HIV 2-LTR circleDNA molecule using an alkaline lysis method.
 21. A method of claim 3,wherein the primers used for PCR comprise a (−) strand primer spanningnucleotides 9591 to 9610 of the HXB2 strain of HIV-1, and a (+) strandprimer spanning nucleotides 9650-9669 of the HXB2 strain of HIV-1.
 22. Amethod of treatment for HIV infection in a mammal, the method comprisingadministering to the mammal one or more anti-HIV agents in an amounteffective to reduce an HIV viral load in the mammal; and detectingHIV-infected cells in the mammal using the method of claim 1, whereintreatment is continued until the level of HIV-infected cells falls below1 in one million peripheral blood mononuclear cell.